Melanoma will strike an estimated 59,000 individuals and cause 7,700 deaths in the United States in 2005, and its incidence is rising faster than that of all other cancers except lung cancer. While primary tumors can be excised surgically, approximately half of all patients develop metastatic disease for which there are few treatment options. Thus, there is an urgent need for new therapies to combat malignant melanoma. Although largely resistant to conventional chemotherapies, malignant melanoma is unusually susceptible to immune therapies. Tyrosinase (TYR) is the prototype melanosomal differentiation antigen and provides an attractive target for vaccine therapy. DNA plasmid vaccines that express TYR have demonstrated promise in preclinical testing and recently entered human testing for melanoma therapy. In this new Phase I project, we utilize a novel and potent viral vector to deliver TYR to the immune system. We will explore vaccine replicon particles (VRP) based on the alphavirus Venezuelan equine encephalitis virus. The VRP platform has elicited potent cellular and humoral immune responses to diverse antigens in a variety of settings, and the technology incorporates layered safety features that render it attractive for use in humans. The overall goal of this project is to determine if our novel TYR-VRP vaccine is superior to an investigational TYR-DNA vaccine in the best available preclinical models of malignant melanoma. The specific aims of this Phase I project are: 1) Identify TYR-VRP dosing regimens that optimally elicit T-cell responses and break immunological tolerance in mice; 2) Compare TYR-VRP and TYR-DNA vaccines for tumor protection in a stringent mouse model of melanoma; and 3) Compare TYR-VRP and TYR-DNA vaccines for the ability to elicit human HLA-A2 restricted T-cell responses in transgenic animals. Project success requires that TYR-VRP demonstrate clear superiority over TYR-DNA in terms of both tumor protection and the generation of HLA-A2 restricted responses. Success in the project would represent an important advance to this field and provide strong impetus for developing TYR-VRP for human testing. [unreadable] [unreadable] [unreadable]